Enzymatic oxidation of Cephalosporin C (Ceph C) to glutaryl-7-aminocephalosporaic acid (glutaryl-7-ACA) has been shown possible on a laboratory scale. (See, for example, Arnold, et al., U.S. Pat. No. 3,821,209, and Fildes et al., U.S. Pat. No. 3,821,209, incorporated herein by reference.) However, a successful and economically feasible scale up to industrial scale production has historically been problematic.
The above conversion is of great commercial importance as it opens up the possibility for an entirely enzymatic route from Cephalosporin C to -amino-cephalosporanic acid (7-ACA), since enzymes which convert glutaryl-7-ACA to 7-ACA are known. [See, for example Shibuya, et al., "Isolation and properties of 7.beta.-(-4-carboxybutanamido)cephalosporanic Acid Acylase-producing Bacteria," Agric. Biol. Chem., 45(7), 1561-1567, 1981]. Further, another advantage of an enzymatic route to 7-ACA is the utilization of an aqueous production mixture, thereby minimizing the hazardous waste solvents generated in the process.
It is known that in order to obtain a good yield of glutaryl-7-ACA, it is essential that all catalase activity be eliminated from the D-aminoacid oxidase. In the above transformation, Cephalosporin C reacts with D-amino acid oxidase to form a thermally unstable .alpha.-keto adipoyl 7-ACA intermediate, which is oxidized in situ by the hydrogen peroxide also produced to the desired glutaryl-7-ACA. However, the naturally-occurring catalase enzyme acts to destroy the hydrogen peroxide. Thus, without a means to deactivate catalase prior to the oxidation, the .alpha.-keto adipoyl intermediate is found to decompose, thereby reducing the yield of desired glutaryl-7-ACA to the desired glutaryl-7-ACA. It is possible to chromatographically remove this contaminant but on industrial scale production, chromatography of this magnitude is not a practical alternative. Thus, the only practical solution is to somehow selectively deactivate the catalase.
Others have reported the possibility of using catalase inhibitors such as ascorbic acid, 3-amino-1,2,4-triazole, sodium cyanide, sodium acetate, sodium formate, sodium fluoride, or sodium azide. Among the above inhibitors, sodium azide is the only one that works well enough to be of practical utility. However, one rather serious drawback to the use of sodium azide, is the formation of a 3-azidomethyl-3-cephem by-product which can be carried over to the final product. This azido contaminant is simply not acceptable in pharmaceutical agents, since the extremely toxic azide ion could possibly be released upon administration to the patient.